Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects 350 million people and kills >600,000 annually. Therapy employs nucleos(t)ide analogs that usually drive HBV to at or below the clinical limit of detection. However, viral replication is not ablated and HBV resurges if drug is withdrawn, so therapy is life-long. Nevertheless, treatment cures up to 6% of patients, so many more patients could be helped by suppressing HBV further. HBV reverse transcription requires the viral ribonuclease H (RNaseH) to destroy the RNA after it has been copied into DNA. Inhibiting the RNaseH blocks viral replication, but anti-RNaseH drugs have not been designed due to technical limitations. We recently developed the first HBV RNaseH inhibitor screening pipeline. We screened >280 compounds containing pharmacophores that inhibit the HIV RNaseH for the ability to block the HBV RNaseH and HBV replication. 41 compounds were N-hydroxyisoquinolinediones (HID), N- hydroxypyridinediones (HPD), or related polyoxygenated heterocycles. Thirteen of these compounds inhibited suppressed viral replication in cells. The most effective HPD/HID compound has an EC50 of 0.57 M against HBV replication in culture (therapeutic index = 26). These compounds chelate Mg++ in the RNaseH active site, as expected from studies on the HIV RNaseH. Here, Drs. Tavis (virologist) and Meyers (medicinal chemist) will conduct an interdisciplinary exploratory R21 project to answer 2 questions needed to generate HPD/HID leads for anti-HBV drug development. Aim 1. What types of modifications to the HPD/HID pharmacophore improve efficacy? We will synthesize a diverse series of ~70 derivatives of the HPD/HID scaffold including predictive characteristics (CLogP, ALOGpS, etc.) in the design and evaluate their efficacy against viral replication. We will assess drug- like properties such as stability in microsomal extracts and hepatocytes for the best compounds. Compound synthesis will be iterative, with results from the initial compounds informing design of later ones. Aim 2. What are the best approaches to maximize selectivity of HPD/HIDs for the HBV RNaseH? Curative anti-HBV therapy is likely to last for many months, so high selectivity for the HBV enzyme and low cytotoxicity will be key. We will counter-screen all compounds against the human RNaseH1 and carefully assess cytotoxicity for the best compounds. Aims 1 and 2 will be conducted concurrently so selectivity and cytotoxicity issues can be addressed during iterative compound design. These R21 studies will generate a structure-activity relationships (SAR) for the HPD/HID pharmacophore that will guide medicinal chemistry optimization, leading to candidate anti-HBV drugs. RNaseH drugs are anticipated to be used in combination with the nucleos(t)ide analogs to suppress HBV replication far enough to clear HBV in many more patients than the current monotherapies can achieve.